GB1587116A - Production of d-amino acid - Google Patents

Description

PATENT SPECIFICATION

( 21) Application No 19963/78 ( 22) Filed ( 31) Convention Application No's 52/067411 52/118928 52/157108 16 May 1978 ( ( 32) Filed 8 Jun 1977 3 Oct 1977 26 Dec 1977 in À Japan (JP)

Complete Specification Published 1 Apr 1981

INT CL 3 C 07 C 99/08 C 07 D 209/20 233/64 317/58 Index at Acceptance C 2 C 1343 1410 1494 20 Y 213 215 220 226 227 22 Y 246 247 251 252 253 306 30 Y 313 31 Y 320 321 326 32 Y 338 341 342 34 Y 35 Y 360 361 362 364 365 366 367 36 Y 370 371 373 37 Y 45 X 45 Y 490 571 620 623 624 626 628 630 634 638 650 652 662 672 747 EA KD KN LA LR LY RL ( 72) Inventors:

SHIGERU NAKAMORI KENZO YOKOZEKI KOJI MITSUGI CHIKAHIKO EGUCHI HISAO IWAGAMI ( 54) PRODUCTION OF D-a-AMINO ACID ( 71) We, AJINOMOTO CO, INC a Japanese company, of No 5-8, Kyobashi 1 chome, Chuo-ku, Tokyo, Japan, do hereby declare the invention, for which we pray that a patent may be granted to us, and the method by which it is to be performed, to be particularly described, in and by the following statement:

This invention relates to a process for producing D-a-amino acids, particularly by an enzymatic process.

D-a-amino acids as a class constitute many compounds and have potential utility as agricultural chemicals or medicinal agents or they can be used as starting materials for these chemicals or medicinal agents For example D-p-hydroxy phenylglycine can be used as a starting material for the antibiotic, amoxycillin.

It is known that D-a-amino acids can be prepared by chemically hydrolyzing 5-substituted hydantoins to DL-a-amino acids, and then separating the D-a-amino acids from the racemic hydrolysis reaction product This method, however, is rather complex in that it requires the separation of D-isomers and racemization of the L-isomer of the amino acids.

It is also known that 5-substituted hydantoins can be enzymatically converted to La-amino acids (Japanese Published Examined Patent Application No 13850/1967) It is further known that the 5-substituted hydantoins can be enzymatically converted to N-carbamyl-D-a-amino acids (Japanese Published Unexamined Patent Application No.

10484/1977, and Proceedings of 1977 Annual Meeting of the Agricultural Chemical Society of Japan p 215 ( 1977)) In the latter process, however, additional steps are required to convert the N-carbamyl-D-a-amino acids to D-a-amino acids.

A need has therefore continued to exist for a simpler and more economic process for obtaining D-a-amino acids.

According to the present invention, there is provided a process for producing a D-a-amino acid, which comprises contacting in an aqueous medium at a p H in the range from 4 to 9 a 5-substituted hydantoin with an effective amount of an enzyme capable of converting the 5-substituted hydantoin to the D-a-amino acid and produced by a microorganism, the microorganism being capable of utilizing the D-isomer of the 5substituted hydantoin as the sole nitrogen source, but substantially incapable of utilizing the Lisomer of the 5-substituted hydantoin as the nitrogen source, and the substituent at the 5-position being such that upon reaction with the enzyme an optionally active D-a-amino acid isomer is produced.

The process of the present invention optionally includes the recovery of the D-a-amino acid which accumulates in the aqueous medium.

It has been found that a wide distribution of bacterial microorganisms exists which produce an enzyme capable of converting 5-substituted-hydantoins to D-a-amino acids These bac( 11) 1 587 116 ( 33) ( 44) À ( 51) ( 52) 1,587,116 teria can especially be found in the genera Pseudomonas, Achromobacter, Alcaligenes, Moraxella, Paracoccus and Arthrobacter The microorganisms can grow in an environment in which the D-isomer of the 5-substituted-hydantoin of this invention is used as the sole nitrogen source, but cannot grow significantly in environments which utilize the L-isomer of the 5-substituted hydantoin as the nitrogen source 5 The bacteria are most readily obtained by the following method:

A sample of microorganisms is inoculated on a screening medium which contains, per deciliter, 0 5 g glucose, 0 01 g yeast extract, 0 1 g KH 2 PO 4, 0 3 g K 2 HPO 4, 50 mg Mg SO 4 À 7 H 20, 1 mg Fe SO 4 À 7 H 20, 1 mg Mn SO 4 À 4 H 20, and 1 0 g of one of the D or L5-substituted-hydantoins of this invention The p H of the screening medium is preferably 10 maintained at a low p H to prevent racemization of the D or L-isomer of the 5-substituted hydantoin The inoculated medium is incubated at 30 C for 1 to 10 days.

Representative specimens of microoganisms which are capable of producing the necessary enzyme are as follows:

Pseudomonas solanacearum AJ 11149 15 (FERM-P 4078, NRRL B-11,255) Pseudomonas caryophylli AJ 11150 (FERM-P 4079, NRRL B-1 i 1,256) Pseudomonas diminuta AJ 11151 (FERM-P 4080, NRRL B 11,257) 20 Pseudomonas diminuta AJ 11152 (FERM-P 4081, NRRLB-11,258) Achromobacter liquefaciens AJ 11198 (FERM-P 4228, NRRLB-11,259) Alcaligenes aquamarinus AJ 11199 25 (FERM-P 4229, NRRL B 11,260) Moraxella nonliquefaciens AJ 11221 (FERM-P 4348, NRRL B- 1,261) Paracoccus denitrificans AJ 11222 (FERM-P 4349, NRRL B 11,262) 30 Arthrobacterfragilus AJ 11223 (FERM-P 4350, NRRL B 1,263) The taxonomic characteristics of the microorganisms mentioned above are as follows: (The experimental methods employed conformed to the methods described in "M J.

Pelczav; Manual of Microgiological Methods", and identifications were made according to 35 "Bergey's Manual of Determinative Bacteriology") AJ 11149 (A) Morphological characteristics 1 Cell form: rods,0 3-0 5 x 1 2-2 gm 2 Pleomorphism: none 40 3 Motility, flagellation: motile, polar flagella 4 Spore: absent Gram stain: negative 6 Acid fast: negative (B) Culture characteristics 45 1) Nutrient-agar plate:

Moderate growth, circular, convex to pulvinate, glistening, smooth, entire, amorphous, opaque, viscid, rosy buff.

2) Nutrient-agar slant: 50 Abundant growth, raised, filiform, soluble pigment not formed.

3) Nutrient broth:

Ring, moderate growth, uniformly turbidic, viscid sediment 55 4) Gelatinstab: notliquefied 5) Litmus milk: unchanged (C) Physiological characteristics:

1 Reduction of nitrate: positive 2 Dentrification: positive 60 3 Testofmethylred: negative 4 VP-test: negative Production of indole: negative 6 Production of hydrogen sulfide: negative 65 3 1 587116 3 7) Hydrolysis of starch: negative 8) Utilization citrate: Koser's citrate medium; negative.

Christensen's citrate medium; positive 5 9) Assimilation of inorganic nitrogen:

Nitrate; positive, Ammonium; positive 10) Formation of pigment: notformed 11 Reaction of urease: positive 12 Reaction of oxidase: positive 10 13 Reaction of catalase: positive 14 Growth range: growupto 38 C,donot grow at 39 C, grow at p H 6 to p H 10 15) Aerobiosis: aerobic 15 16 O-Ftest: O 17 Formation of acid and gas from carbohydrates acid gas L-arabinose + D-xylose + 20 D-glucose + D-mannose + D-fructose D-galactose + Maltose + 25 Sucrose + Lactose trehalose + D-sorbitol D-mannitol 30 inositol glycerine + starch raffinose adonitol 35 salicin dulcitol rhamnose 18) Assimilation of carbon sources:

glucose + ethanol 40 fructose + testosterone L-arabinose arginine saccharose + betaine malonic acid salts 19) Nutritional requirement: none 45 20) Arginine dihydrase: none 21 Accumulation of poly-,/-hydroxy-butyric acid: accumulated According to the taxonomic characteristics mentioned above, AJ 11149 is identified as Pseudomonas solanacearum.

AJ 11150 50 (A) Morphological charactistics 1) Cell form: rods, 0 3 " O 5 x 1 5 -3,m 2 Pleomorphism: none 3 Motility, flagellation: motile, polar flagella 4 Spore: absent 55 5) Gram stain: negative 6 Acid fast: negative (B) Culture characteristics 1) Nutrient-agar plate:

abundant growth, circular, convex to pulvinate, 60 entire, smooth, glistening, opaque, amorphous, viscid, milky to buff.

2) Nutrient-agar slant:

abundant growth, raised, filiform to spreading, soluble pigment not formed 65 1.587 116 4 1,587,116 4 3) Nutrient broth:

abundant growth, membranous, uniformly turbidic, viscid sediment 4) Gelatin stab: not liquefied 5) Litmus milk: unchanged (C) Physiological characteristics 1) Reduction of nitrate: positive 2) Denitrification: positive 3) Test of methylred: negative 4) VP-test negative 5) Production of indole: negative 6) Production of hydrogen sulfide: negative 7) Hydrolysis of starch: negative 8) Utilization of citrate: Koser's citra Christensen's citrate medium, positive.

9) Assimilation of inorganic nitrogen:

nitrate, positive; ammonia, positive 10) Formation of pigment: not formed 11 Urease: positive 12 Oxidase: positive 13 Catalase: positive 14 Growth range: grows up to 3 not grow at 4 from p H 6 to 15) Aerobiosis: aerobic 16 O-Ftest: O 17 Formation of acid and gas from carbohydrates:

te medium, positive; 9.2 C, does 2 C, grows at p H 10 acid gas L-arabinose + D-xylose + D-glucose + D-mannose + D-fructose D-galactose maltose sucrose + lactose trehalose D-sorbitol D-mannitol inositol glycerine starch raffinose adonitol salicin dulcitol rhamnose 18) Assimilation of carbon sources:

glucose + D (-) tartarate D-xylose + meso-tartarate D-ribose + adonitol L-rhamnose + 2,3-butyleneglycol + levulinate m-benzoic acid citraconate tryptamine mesaconate a-amylamine arginine + erythritol + betaine + 19) Nutritional requirements: none Arginine dihydrase: none 21 Accumulation of poly-/3-hydroxy-butyric acid: accumulated According to the taxonomic characteristics mentioned above, AJ 11150 is identified with Pseudomonas caryophylli.

1,587,116 1,587,116 AJ 11151 (A) Morphological characteristics 1 Cell form: rods, 0 3 0 4 x 1 22/um 2 Pleomorphism: none 3 Motility, flagellation: motile, polar flagella 4 Spore: absent Gram stain: negative 6 Acid fast: negative (B) Growth on various media 1) Nutrient-agarplate:

moderate growth, circular, convex, entire, smooth, glistening, opaque, amorphous, butyrous, buff to salmon.

2) Nutrient-agar slant:

moderate growth, effused, filiform to spreading, soluble pigment not formed.

3) Nutrient broth:

moderate growth, uniformly turbidic.

4) Gelatin stab: not liquefied 5) Litmus milk: unchanged (C) Physiological characteristics:

1 Reduction of nitrates: positive 2 Dentrification: negative 3 Test of methyl red: negative 4) VP-test: negative 5) Production of indole: negative 6) Production of hydrogen sulfide: negative 7 Hydrolysis of starch: negative 8 Utilization of citrate:

Koser's citrate medium, positive; Christensen's citrate medium, positive 9) Assimilation of inorganic nitrogen:

nitrate, positive; ammonium, positive.

10) Formationofpigment: notformed 11) Urease: negative 12) Oxidase: positive 13) Catalase: positive 14) Growth range: grows up to 36 5 C, does not grow at 30 C, grows at from p H 6 to p H 9.

15) Aerobiosis: aerobic 16 O-Ftest: acid not produced 17 Formation of acid and gas from carbohydrates L-arabinose D-xylose D-glucose D-mannose D-fructose D-galactose maltose sucrose lactose trehalose D-sorbitol D-mannitol inositol glycerine starch raffinose adonitol salicin dulcitol rhamnose acid gas 6 1587,116 6 18) Assimilation of carbon sources glucose cellobiose DL-,3-hydroxybutyrate + L-histidine + 19) Nutritional requirement: 5 biotine, pantothenic acid, vitamin B 12 and cystine.

20) Accumulation of poly-a-hydroxy-butyric acid: accumulated According to the taxonomic characteristics mentioned above, AJ 11151 can be identified as Pseudomonas diminuta 10 AJ 11152 (A) Morphological characteristics 1 Cell form: rods, 0 3 0 4 x 1 22 gm 2 Pleomorphism: none Motility, flagellation: motile, polar flagella 15 4 Spore: absent Gram stain negative 6 Acid fast: negative (B) Growth on various media 1) Nutrient-agar plate: 20 moderate growth, circular, convex, entire, smooth, amorphous, glistening, opaque, butyrous, buff to salmon.

2) Nutrient-agar slant:

moderate growth, effused, filiform soluble pigment not formed 25 3) Nutrient broth: moderate growth Gelatin stab: not liquefied Litmus milk: unchanged (C) Physiological characteristics 1 Reduction of nitrate: positive 30 2 Denitrification: negative 3 Test of methyl red: negative 4 VP Test: negative Production of indole: negative 6 Production of hydrogen 35 sulfide: negative 7 Hydrolysis of starch: negative Utilization of citrate:

Koser's citrate medium, negative; Christensen's citrate medium, negative 40 9) Assimilation of inorganic nitrogen:

nitrate, positive; ammonium, positive.

10) Formation of pigment: not formed 11 Urease: positive 12 Oxidase: positive 45 13 Catalase: positive 14 Growth range: grows up to 38 C, does not grow at 40 C grows at from p H 6 to p H 9 15) Aerobiosis: aerobic 50 16) O-F test: Acid is not produced 17 Formation of acid and gas from carbohydrates:

acid gas L-arabinose D-xylose 55 D-glucose D-mannose D-fructose D-galactose maltose 60 sucrose lactose trehalose D-sorbitol D-mannitol 65 1,587, 16 7 1587116 7 acid gas inositol glycerine starch raffinose adonitol salicin dulcitol rhamnose 18) Assimilation of carbon sources glucose cellobiose DL-f,8-hydroxybutyrate + histidine + 19) Nutritional requirement:

biotine, pantothenic acid, vitamin B 12 and cystine.

20) Accumulation of poly-f,8-hydroxy-butyric acid: accumulated According to the taxonomic characteristics mentioned above, AJ 11152 can be identified as Pseudomonas diminuta.

AJ 11198 (A) Morphological characteristics 1) Cell form: rods,1 2 x 35/g m 2) Pleomorphism: none 3) Motility, flagellation: motile, peritrichrous flagella 4) Spore: absent 5) Gram stain: negative 6) Acid fast: negative (B) Growth on various media' 1) Nutrient-agarplate:

abundant growth, circular, convex to pulvinate, entire, smooth, glistening, opaque, amorphous, viscid, milky to buff.

2) Nutrient agar slant:

abundant growth, effused, filiform, soluble pigment is not formed.

3), Nutrient broth:

abundant growth, 'membranous, uniformly turbidic, viscid sediment.

4) Gelatin stab: liquefied 5) Litmus milk: unchanged (C) Physiological characteristics 1) Reduction of nitrate: positive 2) Dentrification: negative 3) Test of methyl red: negative 4) VP Test negative 5) Production of indole: negative 6) Production of hydrogen sulfide: negative 7) Hydrolysis of starch: negative 8) Utilization of citrate:

Koser's citrate medium, positive; Christensen's citrate medium, positive.

9) Assimilation of inorganic nitrogen:

nitrate, negative; ammonia, negative.

10) Formation of pigment: not formed 11) Urease: negative 12) Oxidase: positive 13) Catalase: positive 14) Growth range: grows at 15 to 35 C, optimum 20 C to 25 C, grows atp H 6 top H 9.

According to the taxonomic characteristics mentioned above, AJ 11198 can be identified as Achromobacter liquefaciens.

AJ 11199 (A) Morphological characteristics 1) Cell form: rods,0 7 x 2-4, m 1.587116 8 1,587,116 8 2) Pleomorphism: none 3)Motility, flagellation: motile, peritrichrous flagella 4 Spore: absent Gram stain: negative 6) Acid fast: negative 5 (B) Culture characteristics 1) Nutrient-agar plate:

moderate growth, circular, convex to pulvinate, smooth, entire, glistening, opaque, butyrous, amorphous, rosy buff to honey 10 2) Nutrient-agar slant:

abundant growth, effused, filiform, soluble pigment not formed.

3) Nutrient broth:

moderate growth, membranous, uniformly turbidic, 15 ring, viscid sediment.

(C) Physiological characteristics:

1 Reduction of nitrates: negative 2 Denitrification: negative 3 Test of methyl red: negative 20 4 VP test: negative 5Production of indole: negative 6 Production of hydrogen sulfide: positive 7) Hydrolysis of starch: positive 25 8) Utilization of citrate:

Koser's citrate medium, positive; Christensen's citrate medium, positive.

9) Assimilation of inorganic nitrogen:

nitrate, negative; ammonium, negative 30 10) Formation of pigment: not formed 11 Urease: positive 12 Oxidase: positive 13 Catalase: positive 14 Growth range: grows at from 15 to 40 C, 35 optimum: 20 to 25 C, grows at from p H 6 to p H 10 15) Aerobiosis: aerobic 16) O-F test: both O and F negative 17) Formation of acid and gas from carbohydrates 40 acid gas L-arabinose + D-xylose + D-glucose + D-mannose + 45 D-fructose + D-galactose + maltose sucrose lactose 50 trehalose D-sorbitol + (weak) D-mannitol + (weak) inositol + (weak) glycerine + (weak) 55 18) G Ccontentin DNA 58 0 % According to the taxonomic characteristics mentioned above, AJ 11199 can be identified as Alcaligenes aquamarinus.

AJ 11221 (A) Morphological characteristics 60 1) Cellform: plump to short rods, 0.4 1 x 1 2 5 gm 2) Pleomorphism: none 3 Motility: none 4 Spore: absent 65 1,587,116 5) Gram stain: negative 6) Acid fast: negative (B) Culture characteristics 1) Nutrient-agar plate:

moderate growth, circular, convex to be raised, entire, opaque, glistening, amorphous, smooth, white gray 2) Nutrient agar slant:

moderate growth, effused, filiform 3) Nutrient broth: uniformly 4) Gelatin stab: not liquef 5) Litmus-milk: not liquef (C) Physiological characteristics 1) Reduction of nitrates:

2) Dentrification:

3) MR-test:

4) VP-test:

5) Production of indole:

6) Production of hydrogen sulfide:

7) Hydrolysis of starch:

8) Utilization of citrate:

9) 10) 11) 12) 13) 14) 15) 16) 17) 18) 19) 20) 21) 22) 23) 24) 25) Christensen's citrate medium, positi' Assimilation of inorganic nitrogen:

nitrate, positive; ammonium, positiv Formation of pigment:

Urease:

Oxidase:

Catalase:

Growth range:

Aeribiosis:

O-F test:

Formation of acid and gas from carb turbid ied ied, slightly alkalified positive negative negative negative negative negative negative Koser's citrate medium, negative; ve.

e.

not formed negative positive positive grows up to 37 C, grows at from p H 6 to p H 9.

aerobic 0 )hydrates:

acid L-arabinose + (we D-xylose + (w, D-glucose + (w, D-mannose D-fructose + (w, D-galactose maltose sucrose lactose starch trehalose D-sorbitol D-mannitol inositol glycerol Oxidation of gluconic acid (Haynes' method):

negative Utilization of malonic acid (Ewing et al's method): negative Deaminase of phenylalanine (Ewing et al's method): negative Decarboxylase (M 011 er's method):

lysine ornithine arginine Arginine dihydrase (Stanier et al's method) negative Hydrolysis of casein: negative Hydrolysis of DNA: negative Accumulation of poly-/g-hydroxybutyrate: negative eak) eak) eak) eak) gas -0 1 -5 av 1 10 According to the taxonomic characteristics mentioned above, AJ 11221 can be identified as Moraxella nonliqeufaciens.

AJ 11222 (A) Morphological characteristics 1) Cellform: plump to short rods, 0.4 lx 12) Pleomorphism: none 3 Motility, flagellation: none 4 Spore: absent 5) Gram stain: negative 6) Acid fast: negative (B) Culture characteristics 1) Nutrient-agar plate:

moderate growth, circular, convex to raised, smooth, entire, glistening, opaque, butyrous, amorphous, buff to milky.

2) Nutrient-agar slant:

moderate growth, effused, filiform.

3) Nutrient broth: uniformlyt 4) Gelatin stab: not liquefie 5) Litmus milk: not liquefie a (C) Physiological characteristics 1) Reduction of nitrates: p 2) Denitrification: n 3) MR-test: n 4) VP-test: n 5) Production of indole: n 6) Production of hydrogen sulfide: n 7) Hydrolysis of starch: n 8) Utilization of citrate: < Christensen's citrate medium, positive.

9) Assimilation of inorganic nitrogen:

nitrate, positive; ammonium, positive.

10) 11 12 13) 14) 15) 16) 17) 18) 19) 20) lkalified ositive egative egative egative egative 2.5 p, urbidic ed ed, slightly egative egative loser's citrate medium, negative; Formation of pigment: not formed Urease: negative Oxidase: positive Catalase: positive Growth range: grows up to 36 C, grows at from p H 6 to p H 10.

Aerobiosis: aerobic O-F test: O Formation of acid and gas from carbohydrates acid L-arabinose + (weak) D-xylose + (weak) D-glucose + (weak) D-mannose D-fructose + (weak) D-galactose maltose sucrose lactose trehalose D-sorbitol D-mannitol inositol glycerine starch Oxidation of gluconic acid (Haynes' method):

negative Utilization of malonic acid (Ewing et al's method): negative Deamination of phenylalanine (Ewing et al's gas 1.587 116 11 5711 11 method): negative 21) Decarboxylase (M 011 er'smethod):

lysine arginine ornithine 22) Arginine dihydrase (Stanier et al's negative method):

23) Decomposition of casein: negative 24) Decomposition of DNA: negative Accumulation of poly-,/-hydroxybutyrate: accumulated (D) GC-contentin DNA: 65 4 % According to the taxonomic characteristics mentioned above, AJ 11222 can be identified as Paracoccus denitrifications.

AJ 11223 (A) Morphological characteristics 1) Cell form: rods,0 5 0 9 x 1 2 5 0 am 2) Pleomorphism: none 3) Motility, flagellation: motile, peritrichrous flagella 4) Spore: absent 5) Gram stain: weakly positive 6) Acid fast: negative (B) Culture characteristics 1) Nutrient-agar plate:

moderate growth, circular, convex to unbonate, entire to undulate amorphous, dull, rough, buff to straw.

2) Nutrient-agar slant:

moderate growth, effused to raised, filiform to spreading.

3) Nutrient broth: ring, not turbidi 4) Gelatin stab: not liquefied 5) Litmus-milk: not liquefied, sli alkalified (C) Physiological characteristics 1) Reduction of nitrates:

2) Denitrification:

3) MR-test:

4) VP-test:

5) Production of indole:

6) Production of hydrogen sulfide:

7) Hydrolysis of starch:

8) Utilization of citrate:

10) 11) 12) 13) 14) 15) 16) 17) Christensen's citrate medium, negative.

Assimilation of inorganic nitrogen:

nitrate, positive; ammonium, positive.

Formation of pigment:

Urease:

Oxidase:

Catalase:

Growth range:

Aerobiosis:

O-F test:

Formation of acid and gas from carbob L-arabinose D-xylose D-glucose D-mannose D-fructose D-galactose maltose ghtly positive negative negative negative negative negative negative Koser's citrate medium, negative; not formed negative positive positive grows up to 37 C, grows at from p H 6 to p H 9.

aerobic iydrates acid + (weak) + (weak) + (weak) + (weak) + (weak) gas 1.587116 1.587,116 2 2.

2 ' acid sucrose + (weak) lactose trehalose D-sorbitol D-mannitol + (weak) inositol + (weak) glycerine + (weak) starch 8) Oxidation of gluconic acid (Haynes' negative method):

9) Utilization of malonic acid (Ewing et al's negative method):

0) Deamination of phenylalanine (Ewing et al's negative method):

1) Decarboxylase (M 011 er's method):

lysine ornithine arginine 2) Arginine dihydrase (Stanier et al's method): negative 3) Decomposition of casein: negative 4) Decomposition of DNA: negative 5) Accumulation ofpoly-f 3-hydroxybutyricacid: not accumulated 6) Nutritional requirement requiring biotine 7) Assimilation of carbohydrates (Stainer's method):

D-glucose + saccarate trehalose + propionate 2-keto-gluconate butyrate m-inositol + adonitol L-valine propylene-glycol f,-alanine + ethanol DL-arginine + D-xylose betaine + D-ribose L-arabinose + L-rhamnose saccrose + "polysorbate 80 " levulinate malonate citraconate testosterone meso-tartarate cellobiose D(-) tartarate DL-/g-hydroxybutyrate sorbitol + L-histidine mesaconate pantothenic acid erythritol acetate 2,3-butylene-glycol succinate m-benzoic acid citrate p-benzoic acid L-ornithine tryptamine 5-keto-gluconate a-amyl amine L-lysine DL-lactic acid + L-alanine D-fructose + dulcitol 8) GC-content in DNA: 60 6 % ) Di-basicamino acid in cell-wall: lvsine gas + + + + + + + According to the microbiological characteristics mentioned above, A J 11223 belongs to the genus Arthrobacter AJ 11223 cannot be identified with a known species of the genus Arthrobacter from the point of view of the amino acid pattern in the cellwall, the gelatin decomposition, the motility, the vitamin-requirement, and the starchdecomposition Thus, AJ 11223 can be considered as a novel species of Arthrobacter, and designated as Arthrobacterfragilus.

As far as the 5-substituted hydantoin starting material is concerned, very many hydantoins can be converted to D-a-amino acids by the method of this invention, and therefore, a very wide variety of 5-substituted hydantoins may be used in this invention The 5-substituent, of course, should be chosen such that upon reaction with the enzyme, an optically active D-a-amino acid isomer is produced Suitable 5-substituent group for 5substituted hydantoins include:

1 Straight, branched or cyclic saturated aliphatic hydrocarbon radicals, examples being:

28 1.587,116 methyl, ethyl, propyl, iso-propyl, iso-butyl,l-methyl-propyl, tert-butyl, cyclo-hexyl, and cyclo-pentyl radicals.

2 Straight, branched, or cyclic unsaturated aliphatic hydrocarbon radicals, examples being:

2-propenyl, 1-cyclohexenyl, and 1,4-cyclohexadienyl radicals.

3 Straight, branched or cyclic saturated or unsaturated aliphatic'hydrocarbons radicals in 5 which one or more hydrogen atom is substituted with one or more substituent, of which examples include:

hydroxyl, carboxyl, sulfhydryl, alkylmercapto, amino, alkylamino, alkoxy, carbamoyl, guanidino, ureido, sulfoxyl, nitro, halogeno, acyl, amino-sulfenyl, arylmercapto, 4-imidazolyl, and 4-thienyl radicals 10 4 Aromatic hydrocarbon radicals, examples being:

phenyl and naphthyl radicals.

Aromatic hydrocarbon radicals in which one or more hydrogen atom is substituted with one or more substituents, of which examples include:

alkyl, alkenyl, cyclic alkyl or alkenyl, hydroxyl, alkoxyl, halogeno, benzyloxy, 15 benzyloxy-methyloxy, methoxy-methyloxy, acyloxy, acyl, aryloxy, aminosulfenyl, trifluoro-methyl, alkylmercapto, amino, acylamino, alkylamino, nitro, carboxyl, and carbamoyl radicals.

6 Heterocyclic radicals, of which examples include:

2-thienyl, 5-thiazole, 4-imidazole, and 2-furyl radicals 20 7 Heterocyclic radicals in which one or more hydrogen atom is substituted with one or more substituents, of which examples include those listed under " 5 " above.

8 Aralkyl and substituted aralkyl radicals.

When the microorganisms as mentioned above are cultured in conventional culture media, an enzyme capable of converting the hydantoin compounds to D-a-amino acids is produced 25 mainly in the cells of the microorganisms and is present to a slight extent in the supernatant of the cultured liquid.

The culture media used can contain carbon sources, nitrogen sources, inorganic ions, and when required, minor organic nutrients When the culture media contain small amounts of the 5-substituted hydantoins, the enzyme activity produced is generally much higher 30 Suitable carbon sources include, for example, carbohydrates such as glucose, and sucrose; organic acids such as acetic acid; alcohols such as ethylalcohol, and hydrocarbons Suitable nitrogen sources include, for example, ammonia and urea Suitable providers of inorganic ions include, for example, phosphates and magnesium, ferrous, calcium, and potassium salts.

Suitable organic nutrients include, for example, vitamins, amino acids and crude materials 35 containing such organic nutrients as yeast extract, peptone, bouillon, or corn steep liquor.

Cultivation is carried out at a p H of from 4 to 8 and preferably at a temperature in the range from 25 to 40 C under aerobic conditions After a one-half to 2 days of cultivation, the enzyme is chiefly produced in the cells Accordingly, a culture broth containing cells and intact cells can be used as the enzyme source Moreover, a homogenate of cells, cells treated 40 with supersonic waves, freeze-dried cells or cells dried with organic solvents such as acetone may also be used as the enzyme source The protein fraction separated, for example, from the homogenate of cells or from the sonicate of cells, by a conventional method such as gelfiltration or the salting-out method can be also used as the enzyme source Cells or other enzyme sources as above can be used after immobilization It is expected that there is more 45than one enzyme participating in the conversion of the 5-substituted hydantoins to the D-a-amino acids.

The conversion of the 5-substituted-hydantoins to D-a-amino acids can be carried out by contacting the 5-substituted hydantoins with the cells of the microorganisms of this invention in the culture media during the course of the cultivation of the microorganisms, or by 50 contacting 5-substituted hydantoin with the cells or the enzyme sources as mentioned above in reaction media When the 5-substituted hydantoin is contacted with the cells in culture media, the hydantoin is preferably added after the microorganism has partly or wholly propagated In order to avoid inhibition of the growth of the microorganism by high concentrations of the hydantoin, the concentration of the hydantoin in the culture media 55 should preferably be maintained at a low level by intermittently feeding small portions of the hydantoin to the culture media The cultivation is continued until no further hydantoin is effectively converted to D-a-amino acids.

The reaction media in which the 5-substituted hydantoin is contacted with the cells or the enzyme sources may optionally contain anti-oxidants, surface active agents, coenzymes 60 and/or hydroxylamine to improve the yield of D-a-amino acid The reaction media are maintained preferably at a temperature from 10 to 70 C, and at a p H of 4 to 9 to 100 hours.

The D-a-amino acid which accumulates in the culture media or the reaction media can be recovered very easily in a conventional manner such as by using ion exchange resins, or precipitation of the amino acid product at its iso-electric point, since very little L-isomer of 65 1.587 116 the D-a-amino acid product is produced.

Having generally described this invention, a further understanding can be obtained by reference to certain specific examples which are provided herein for purposes of illustration only, and are not intended to be limiting unless otherwise specified.

Quantitative analysis of D-a-amino acid in the following examples was carried out as 5 follows: The amino acid was developed on filter paper (solvent; n-butanol 2 parts, acetic acid 1 part and water 1 part), and the paper was sprayed with ninhydrin solution The colordevelopment was measured at 570 m/,, after extraction with 50 % ethanol Quantitative analyses were conducted by liquid-chromatography The D and L-isomer contents were determined by measuring the optical rotation of the product 10 Example 1

An aqueous culture medium of p H 7 0 was prepared which contained, per deciliter, 0 5 g glucose, 0 5 g (NH 4)2504, O 1 g KH 2 PO 4, O lg K 2 HPO 4, 0 05 g Mg SO 4 7 H 20, 1 mg Fe SO 4 7 H 20, lmg Mn SO 4 4 H 20, 1 O g yeast extracts and 0 2 g DL-5methylmercaptoethyl hydantoin Fifty ml batches of the aqueous culture medium were placed in 500 ml flasks which were 15 shaken and heated to sterilize at 120 C for 15 minutes Pseudomonascaryophylli AJ 11150, which was previously cultured on bouillon-agar slants at 30 C for 24 hours, as a one-loopful inoculum was transferred from the bouillon-agar slants into each batch of the aqueous culture medium Cultivation was carried out at 30 C with shaking for 20 hours The cells produced in the culture liquid were collected by centrifugation and washed with the same volume of 20 physiological saline as the culture liquid The cells thus obtained ( 5 g/dl) were suspended in samples of 0 1 M phosphate-buffer (p H 8 0) each containing 1 g/dl of the 5substituted-hydantoins shown in Table 1 (final volume being 5 ml) Each reaction mixture was held at 30 C for 16 hours.

The amounts of the D-a-amino acids produced in the reaction mixtures and the optical 25 rotation of the amino acids were determined by the method mentioned before Prior to the determination, cysteine and homocysteine in the culture liquid were oxidized to cystine and homocystine The results are shown in Table 1.

Table 1

Opticl rottion f la 120 Optcalrottio of la D the amino authentic 5-substituted-hydantoin used D-a-amino acid formed acid formed sample conditionsDL-5-methyl hydantoin D-alanine 2 16 mg/mi -14 30 14 70 C= 1 6 N-HCI DL-5-isopropyl hydantoin D-valine 0 70 -27 80 27 90 C= 0 8 DL-5-isobutyl hydantoin D-leucine 0 56 -15 20 15 20 C= 0 4 DL-5-( 1 '-methylpropyl) hydantoin D-isoleucine 1 21 -38 20 4070-1 DL-5-hydroxymethyl hydantoin D-serine 3 91 -14 10 14 60 C= 1 2 N-HCI DL-5-( 1 '-hydroxyethyl) hydantoin D-threonine 2 82 -27 40 28 50 C= 0 6 H 20 DL-5-sulfhydrylmnethyl hydantoin D-cysteine + D-cystine 4 84 + 220 70 + 222 50 C= 0 2 1 N-WIDL-5-methylmercaproethyl hydantoin D-methionine 5 38 23 80 24 10 C= 0 8 6 N-HCI DL-5-carbamoyimethyl hydantoin D-asparagine 4 82 -30 80 31 30 C = 0 2 3 NHCI DJL-5-carbamoylethyl hydantoin D-glutamine 4 26 -6 70 6 60 C= 0 4 H 20 DL-5-benzyl hydantoin D-phenylalanine 2 86 + 32 30 + 3430 C= 0 2 H,0 DL-5-(p-hydroxybenzy I) hydantoin D-tyrosine 2 02 + 11 60 + 11 80 C= 0 5 1 N-HCI DL-5-indolylmethyl hydantoin D-tryptophan 1 86 + 30 90 + 32 50 C= 0 1 H 20 DL-5-( 5 '-hydroxyindolylmethyl) hydantoin D-5-hydroxytryptophan 0 92 -15 60 16 20 C= 1 4 N-HC 1 DL-5-carhoxymethyl hydantoin D-aspartic acid 0 62 -26 20 26 20 C = 0 8 6 N-HCI DL-5-carboxyethyl hydantoin D-glutamic acid 0 56 -30 60 32 00 C= 1 2 NHCI DL-5-( 4 '-imidazoylmethyl) hydantoin D-histidine 2 05 -12 20 12 40 C= 0 1 6 N-H Gl DL-5-( 4 '-aminobutyl) hydantoin D-lysine 1 02 -21 80 22 50 C= 0 8 DL-5-( 3 '-guanidopropyl) hydantoin D-arginine 0 69 -26 20 27 20 1 DJL-5-( 3 '-aminopropyl) hydantoin D-ornithine 0 93 -24 20 25 80 C= 0 4 D 1-5-( 3 '-ureidopropyl) hydantoin D-citruline 1 14 -24 60 25 50 C= 0 8 DL-5-( 3 '-sulfhydrylethyl) hydantoin D-homocystein 3 83 -76 90 79 20 C= 0 5 5 N-HCI DL-5-phenyl hydantoin D-phenylglycine 3 94 -151 7 -155 00 C= 1 1 N-HI DL-5-(p-hydroxyphenyl) hydantoin D-p-hydroxyphenylglycine 4 50 -158 50 161 2 ' DL-5-(p-chlorophenyl) hydantoin D-p-chlorophenylglycine 2 52 136 9 ' -138 6 ' DL-5-(p-methylphenyl) hydantoin D-p-methylphenylglycine 1 14 -147 10 149 20 DL-5-(p-methoxyphenyl) hydantoin D-p-methoxyphenylglycine 4 39 -154 1 ' 453 80 DL-5-(p-henzyloxyphenyl) hydantoin D-p-benzyloxyphenylglycine 0 92 142 7 ' -147 6 ' DL-5-( 3 ',4 '-dihydroxybenzyl) hydantoin D-3,4-dihydroxyphenylalanine 0 87 + 10 90 + 11 70 DL-5-( 3 ',4 '-dimethoxybenzyl) hydantoin D-3,4-dimethoxyphenylalanine 0 56 + 10 00 + 10 30 DL-5-( 3 ' 4 '-methylenedioxybenzyl) hydantoin D-3,4methylenedioxyphenylalanine 1 25 8 70 + 9 10 (.11 Co O 16 1,587 116 16 Example 2

Achromobacter liquefaciens AJ 11198 and Alcaligenes aquamarinus AJ 11199 were cultured in the same manner as shown in Example 1 in an aqueous culture medium containing, per deciliter, 2 g glucose, O 5 g (NH 4)2504, O lg KH 2 PO 4, O lg K 2 HPO 4, 0 05 g Mg SO 4 7 H 20, lmg Fe SO 4 7 H 20, lmg Mn SO 4 À 4 H 20, 1 Og yeast extract, O 2 g DL-5 5 methylmercaptoethyl hydantoin and 4 O g Ca CO 3 (separately sterilized), at p H 7 0 The cells thus obtained were contacted in 1 0 M phosphate buffer with the 5substituted-hydantoins shown in Table 2 in the same manner as described in Example 1 The results are shown in Table 2.

Table 2

AJ 11198 AJ 11199 Optical rotation of.

Amount of Amount of authentic Measurement condition D-a-amino Optical D-a-amino Optical D-a-amino Of optical rotation 5-substituted-hydantoin used D-a-amino acid formed acid formed rotation acid formed rotation acid la lbi" DL-5-methyl hydantoin D-alanine 0 82 mg/dl 14 40 -3 08 mg/mi 14 60 14 70 C= 1 6 N-HCI DL-5 itoin D-valine 0 38 27 6 3 89 26 40 27 90 C= 0 8 DL-5 isopropylhydan n D-leucine 0 32 15 3 0 76 15 00 15 21 C= O 4 DL-5-isobuthylpranoinl hdnti D-isoleucine 0 68 38 9 1 42 4010 407 DL-5 (I-hyrxmethylrpyantomf D-serine 1 92 14 5 3 86 -14 30 146 C= 1 1 NHIC 1 DL-5-(I'-hydroyetbl hdtoin D-threoninle 1 49 27 9 2 91 27 60 285 C= 0 6 H 20 DL-5-,sutfhydry cht hdatin D-cysteine +D-cystine 2 21 + 221 6 5 20 + 22210 + 2225 C= 0 2 IN-HCI DL-5-ethym othylhydatoin D-methionine 4 04 238 5 96 24 80 241 ' C= 0 8 6 N-H Cl DL-5-carbamoyletyl hydantoin D-asparagine 2 67 31 0 5 02 313 31 3 C= 0 2 3 N-HCI DL-5-cabmetylerapoehyydantoif D-glutamine 3 47 6 4 4 56 O 6 C O 2 DL-5-cabenylethyda ydaftoif D-phenylalanine 2 47 + 33 8 3 81 + 33 60 + 343 C= 0 2 DL-5-phyrxbenzy Ilhydantoin D-tyrosine 1 28 + 11 6 2 03 + 10 90 + 11 8 ' C= 0 5 1 N-HCI DL-5-(inhdrolybmetyl hydantoin D-tryptophan 1 41 + 31 8 2 64 + 31 60 + 325 C= 0 1 1120 DL-5-( 5 '-hydroxyindolylmethyl) hydantoifl D-5 hydroxytryptophan 0 49 15 4 0 82 1610 162 C= 1 4 N-HC 1 DL-5-carboxymethyl hydantoifl D-aspartic acid 0 54 26 3 0 71 25 90 262 C= 0 8 6 N-HIC 1 DL-5-carboxyethyl hydantoin D-glutamicacid 0 40 30 9 0 86 31 80 320 C= 1 2 N-FIC 1 DL-5-( 4 'imidazoylmnethyl) hydantoin D-histidine 1 09 12 2 2 52 11 9 9 12 4 ' Coo O 1 6 N-HCI DI-5 -amnbty)hdati-lysine 1 96 22 6 2 19 224 ' 22 50 C= 0 8 DI 'Yaminopopuyl) hydantoin D-arginine 0 24 26 4 10 7 72 DI-5 ( 3 '-uamndopropyl) hydantoin D-ornithine 0 81 24 7 1 21 25 2 2 25 80 C= 0 4 D 5 ( 3 '-urcidopropyl) hydantoifl D-citruiline O 6-252 136-2 3 2550 C= O 58 N-C D 5 ( 3-sulfhydrylethyl) hydantoin D-homocystein 2 04 77 1 4 23 78 17 O C 055-C DI 5 phenyl hydantoin D-phenylglycine 4 12 -152 0 4 27 -1531 ' 155 00 Cool 1 N-HCI DL-5-(p-hydroxyphernyl) hydanstoif D-p-hydroxyphenylglycine 3 19 159 1 5 26 1600 O 161 20 DI-5-(p-chlorophenyl) hydantoin D-p-chlorophenylglycine 2 30 -137 2 2 35 1387 ' 138 60 D 1-5-(p-inethylphenyl) hydanon D-p-nmethylphentylglycine 2 09 -149 1 2 26 1449 -149 20 DL-5-(-ethoxyphnl hyatoin D-p-methoxyphenylglycine 4 58 -151 9 4 94 154 10 -153 80 òL-5-p-benzylxyphnl anltoin D-p-benzyloxyphenylglycine 0 47-161 3 471 470 òL-5 ( 3 ' '-dihydroxybnyydantoin D-3,4-dihydroxyphenylalanine 0 42 + 10 7 1 24 + 11 00 + 11 70 òL.5-{ 3 ',4 '-dimethoxyen ylhdantoin D-3,4-dimethoxyphenylalanine 0 35 + 10 1 0 98 + 10 00 + 10 30 DL 5-(',4 '-methylen dioxybenzyl) hydantoiflD-3,4methylenedioxyphenylalanine 0 92 + 8 7 1 66 + 9 20 91 DL 5-cyanoethyl hydantoin D-cyanoethylglycine 4 02 + 27 2 5 42 + 26 90 + 28 00 12 J 1 -C 1 18 1 587116 18 Example 3

An aqueous culture medium was prepared such that it contained, per deciliter, 2 O g glucose, 0 5 g (NH 4)2 SO 4, 0 1 g KH 2 PO 4, 0 1 g K 2 HPO 4, 0 05 g Mg SO 4 7 H 20, lmg Fe SO 4 7 H 20, lmg Mn SO 4, 1 O g yeast extract, 1 O g peptone, O 2 g DL5methylmercaptoethyl hydantoin and 4 O g Ca CO 3 (separately sterilized) and was adjusted to 5 p H 7 0 Fifty ml batches of the aqueous culture medium were placed in 500 ml flasks and were heated to sterilize the same at 120 C for 15 minutes.

Moraxella nonliquefaciens AJ 1221, Paracoccus denitrificans AJ 11222, and Arthrobacter fragilus A J 11223 were cultured on bouillon-agar slants at 30 C for 30 hours One loopful of inocula of each of the microorganisms was transferred from the bouillonagar slants into 10 samples of the aqueous culture medium mentioned above A second cultivation was carried out at 30 C for 20 hours while the flasks were shaken The cells in the culture liquids thus obtained were collected by centrifugation, and washed with the same volume of physiological saline as the culture liquid The 5-substituted-hydantoins listed in Table 3 were converted to D-a-amino acids in the same manner as described in Example 1 The results are shown in 15 Table 3.

Moreover, the amino acids which were formed were isolated and purified, and were ascertained as the D-form by optical rotation measurements.

Example 4

Pseudomonas solanacearum AJ 11149, Pseudomonas diminuta AJ 11151 and 20 Pseudomonas diminuta AJ 11152 were used in place of Pseudomonas caryophlli AJ 11150 in repetitions of the procedure of Example 1 The results are shown in Table 4.

1.587,116 Table 3

Amount of D-az-amino acid formed (mg/mi) 5-substituted-hydantoin used D-a-aminoacidformed AJ 11221 AJ 11222 AJ 11223 DL-5-methyl hydantoin D-alanine 3 09 0 33 2 94 DL-5 _isopropyl hydantoin D-valine 3 92 0 59 3 62 DL-5-isobutyl hydantoin D-Ieucine 1 56 0 26 0 81 DL-5-( 1 '-methylpropyl) hydantoin D-isoleucine 4 29 0 24 1 03 DL-5-hydroxymethyl hydantoi'n D-serine 3 52 2 81 3 50 DL-5-( 1 '-hydroxyethyl) hydantoin D-threonine 5 16 2 04 2 51 DL.5-sulfhydrylmethyl hydantoin D-cysteine + D-cystine 5 86 3 00 4 90 DL-5-methylmercaptoethyl hydantoin D-methionine 6 92 3 12 5 56 DL-5-carbanioylmethyl hydantoin D-asparagine 5 19 3 82 4 94 DL-5-carbamoylethyl hydantoin D-glutamine 4 72 4 10 4 07 DL-5-benzyl hydantoin D-phenylalanine 3 41 1 26 3 61 DL-5-(p-hydroxybenzy I) hydantoin D-tyrosine 2 43 1 62 1 95 DL-5-indolylmethyl hydantoin D-tryptophan 2 82 1 02 2 23 DL-5-( 5 '-hydroxyindorylmethyl) hydantoin D-5-hydroxytryptophan 0 97 0 53 0 69 DL-5-carboxymethyl hydantoin D-aspartic acid 0 72 0 49 0 68 DL-5-carboxyethyl hydantoin D-glutamic acid 1 68 0 38 0 72 DL:55 J( 44 '-iidazoylmethyl)hydantoin D-histidine 2 92 1 68 2 14 DL-5 ( 4-minobutyl) hydantoin D-lysine 1 23 0 52 1 92 DL-5-( 3 '-guanidopropyl) hydantoin D-arginine 1 21 0 62 1 07 DL.5-( 3 ' ainoroy) hydantoin D-ornithine 1 42 0 91 1 28 DL-5-( 3 ':m-urindoopproppyl) hydantoin D-citruline 1 42 0 78 1 38 DL-5-( 3 '-sulfhydrylethyl) hydantoin D-hornocystein 5 03 3 92 4 13 DL-5-phenyl hydantoin D-phenylglycine 6 26 3 00 4 09 DL-5 phydroxyphienyl) hydantoin D-p-hydroxyphenylglycine 6 34 3 02 5 00 DL-5 pchlorophenyl) hydantoin D-p-chlorophenylglycine 3 14 2 96 2 39 DL-5 pmethylphenyl) hydantoin D-p-methylphenylglycine 2 97 3 01 2 30 DL-5 pmethoxyplhenyl) hydantoin D-p-methoxyphenylglycine 5 09 3 06 4 98 DL-5 pbenzyloxyphenyl) hydantoin D-p-benzyloxyphenylglycine 2 09 0 92 1 09 DL-5 3 '4-dihydroxybenzyl) hydantoin D-3,4-dihydrooxyphenylalanine 1 24 0 87 1 22 DL 5 3 '4 '-dimethoxybenzyi) hydantoin D-3,4-dimethoxyphenylalanine 0 98 0 42 0 96 DL 5 3 ' 4 '-methylendioxybenzyl) hydantoin D-3,4methylenedioxyphenylalanine 2 06 0 95 1 73 DL-5-cyanoethyl hydantoin D-cyanoethylglycine 6 34 3 47 5 63 DL-5-( 1 '-sulfhydrylI'-mnethylethyl) D-penicillamine 3 92 1 46 2 92 U, :-.

Table 4

5-substituted-hydantoin used DL-5-methyl hydantoin DL-5 isopropyl hydantoin DL-5-isobutyl hydantoin DL-5-( 1 I'-methyipropyl) hydantoin DL-5-hydroxymcthyl hydantoin DL-5-( I -hydroxycthyl) hydantoin DL-5-sulfhydrylmethyl hydantoin DL- 5-methylmercaptoethyl hydantoin DL-5-carbamoylmethyl hydantoin DL-5-carbamoylethyl hydantoin DL-S-bcnzyl hydantoin DL-5 (p-hydroxybenzy I) hydantoin DL-5-indolylmethyl hydantoin DL-5-( 5 '-hydroxyindolylmethyl) hydantoin DL-5-carboxymethyl hydantoin DL-5-carboxyethyl hydantoin DL-5 4 '-imidazoylmethyl) hydantoin DL-5 4 '-aminobutyl) hydantoin DL-5 Y-guanidopropyl) hydantoin DLS-5 3-aminopropyl) hydantoin DL-5 3 '-urcidopropyl) hydantoin DL-5 3 '-sulfhydrylethyl) hydantoin DL-5-phenyl hydantoin DL-5-(p-hydroxyphenyl) hydantoin DL-5-(p-chlorophenyl) hydantoin DL-5-(p-methylphenyl) hydantoin DL-5-(p-methoxyphenyl) hydantoin 3 L (bndziylyoxyphenyl) hydantoin DL 5-( 4-iyroxybenzy I) hydantoin DL-5-(Y 3 '4 '-dimethoxybenzy I) hydantoin DL-5-( 3 ' 4 '-methylencdioxybcnzyl) hydantoin D-a-amino acid formed D-alanine D-valine D-lcucine D-isoleucine D-serine D-threonine D-cysteine + D-cystine D-methionine D-asparagine D-glutamine D-phenylalanine D-tyrosine D-tryptophan D-5-hydroxytryptophan D-aspartic acid D-glutamic acid D-histidine D-lysinc D-arginine D-ornithine D-citruline D-homocystein D-phenylglycine D-p-hydroxyphenylglycine D-p-chlorophenylglycine D-p-methylphenylglycine D-methoxyphenylglycine D-p-benzyloxyphenylglycine D-3,4-dihydroxyphenylalanine D-3,4-dimethoxyphenylalanine D-3,4-methylenedioxyphenylalanine Amount of D-armino acids formed (mg/mi) AJ-11149 AJ-11151 AJ-11152 1.31 0.56 0.42 0.87 3.77 2.58 4.61 4.93 4.02 3.91 2.58 2.52 1.67 0.76 0.46 0.37 1.76 0.84 0.57 0.82 0.89 3.54 4.11 3.62 2.03 0.89 3.81 0.82 1.02 0.62 1.18 2.24 0.70 0.48 1.12 3.86 2.93 5.10 5.72 4.47 4.35 3.81 3.91 2.04 0.91 1.38 1.20 2.24 1.23 0.71 1.14 1.03 4.90 5.30 4.11 2.14 1.24 3.79 1.09 0.98 0.51 1.32 0.52 0.58 0.41 0.34 2.86 1.99 3.29 3.23 3.84 3.62 2.22 2.09 1.23 0.82 0.48 0.34 1.72 0.62 0.31 0.38 0.30 2.45 3.62 2.86 1.63 0.78 3.09 0.55 0.76 0.49 0.59 1-.1 0 i 21 1,587 116 21 Example 5

An aqueous culture medium was prepared in a fashion such that it contained, per deciliter, 2.0 g glucose, 0 5 g (NH 4)2504, O 1 g KH 2 PO 4, 0 1 g K 2 HPO 4, 0 05 g Mg SO 4 '7 H 20, lmg Fe SO 4 7 H 20, 1 mg Mn SO 4 '4 H 20, 1 O g yeast extract, 1 O g peptone and 4 O g Ca CO 3 (separately sterilized), and was adjusted to a p H of 7 0 Fifty ml batches of the aqueous culture 5 medium were placed in 500 ml flasks which were heated to sterilize at 120 C for 15 minutes.

Moraxella nonliquefaciens AJ 11221, Paracoccus denitrificans AJ 11222, and Arthrobacterfragilus AJ 11223 were cultured on bouillon-agar slants at 30 C for 24 hours One loopful of inocula of each of the microorganisms was transferred from the bouillon-agar slants into samples of the aqueous culture medium mentioned above Each sample was then cultured at 10 C with agitation After cultivation for 24 hours, 0 2 g/dl of a sterilized 5-substituted hydantoin listed in Table 5 was added to each culture broth and cultivation was conducted at C for an additional 6 hours The cells in the culture broths thus obtained were harvested by centrifugation, and washed with the same volume of physiological saline as the culture broth 15 DL-5-(p-hydroxyphenyl) hydantoin was converted to D-phydroxyphenylglycine in the same manner as shown in Example 1 The results are shown in Table 5.

Moreover, the samples of p-hydroxyphenylglycine which formed were isolated and purified The amino acid samples formed were ascertained as the D-form by measurement of the optical rotationof the samples 20 Table 5

5-substituted hydantion added to D-p-hydroxyphenylglycine culture broth formed (mg/ml) AJ 11221 AJ 11222 AJ 11223 None 0 96 0 42 1 28 hydantoin 0 98 0 49 1 31 DL-5-methyl hydantoin 2 41 1 03 1 68 DL-5-isopropyl hydantoin 2 39 1 09 2 03 DL-5-isobutyl hydantoin 2 21 1 02 1 96 DL-5-( 1 '-methylpropvl)hydantoin 1 96 1 41 1 73 DL-5-hydroxymethyl hydantoin 1 98 1 37 1 97 DL-5-( 1 '-hydroxyethyl) hydantoin 2 03 1 14 2 01 DL-5-methylmercaptoethylhydantoin 7 34 4 03 5 01 DL-5-carbamoylmethyl hydantoin 2 24 1 21 1 97 DL-5-carbamoylethylhydantoin 2 16 1 46 2 02 DL-5-benzyl hydantoin 4 02 2 09 3 96 DL-5-(p-hydroxybenzyl) hydantoin 2 23 1 04 1 91 DL-5-indolylmethyl hvdantoin 6 92 3 28 5 02 DL-5-( 5 '-hydroxyindolylmethyl) hydantoin 1 86 0 90 1 78 DL-5-carboxymethylhvdantoin 1 92 1 34 1 86 DL-5-carboxyethvl hydantoin 1 92 1 26 1 69 DL-5-( 4 '-imidazoylmethyl) hydantoin 1 82 0 96 1 72 DL-5-( 4 '-aminobutyl) hydantoin 2 16 1 06 1 96 DL-5-( 3 '-guanidopropyl) hvdantoin 1 92 0 96 DL-5-( 3 '-aminopropvl) hydantoin 2 01 0 92 1 98 DL-5-( 3 '-ureidopropvl) hydantoin 1 93 0 87 1 69 DL-5-( 3 '-sulfhydrylethyl) hydantoin 2 01 1 05 2 02 DL-5-phenyl hydantoin 4 02 1 42 3 98 DL-5-(p-hydroxxphenyl)hydantoin 612 2 23 4 64 DL-5-(p-chlorophenxl) hvdantoin 5 28 1 58 4 18 DL-5-(p-methylphenyl) hydantoin 4 92 1 48 3 94 DL-5-(p-methoxxphenyl) hydantoin 7 96 2 42 4 89 DL-5-(p-benzyloxyphenvl) hydantoin 2 95 1 04 2 39 DL-5-( 3 ' 4 '-dihydroxybenzyl) hydantoin 2 83 1 01 2 65 DL-5-( 3 ' 4 '-dimethoxybenzyl)hydantoin 2 41 1 21 2 24 DL-5-( 3 ' 4 '-methvlenedioxybenzyl) hydantoin 2 91 1 31 2 67 DL-5-cyanoethyl hydantoin 7 23 3 29 5 20 Example 6

Pseudomonas caryophylli AJ 11150 was cultured in the same manner as described in Example 1 and the resultant culture broth was centifuged to separate cells The cells were washed with physiological saline Twenty-five grams of the cells were suspended in 500 ml of 0.1 M phosphate buffer solution (p H 8 0) which contained 5 g D-5-(phydroxyphenyl) hydantoin or 5 g L-5-(p-hydroxyphenyl) hydantoin Each reaction mixture was held at 30 C for 27 hours Thereafter, each reaction mixture was centrifuged to remove cells and filtered with ultra-filtering membrane p-Hydroxyphenyl glycine in these filtrates was adsorbed on the H= form of a cation exchange resin ("Diaion" SK-1 B) and were eluted with 1 N NH 4 OH.

Crvstals were obtained by cooling after evaporation of the filtrates, and then purified by recrystallization in water-ethanol solution.

Consequently 3 04 g of crystals were obtained from D-5-(p-hydroxyphenyl hydantoin and 3.20 g of crystals from L-5-(p-hvdroxyphenyl) hydantoin.

These crystals were shown to be the same as authentic D-phydroxypphenylglycine by correspondence of the NMR spectra, the Rf values on thin layer chromatograms and paper chromatograms and by optical rotations.

1.587,116 Example 7

Achromobacter liquefaciens AJ 11198 and Alcaligenes aquamarinus AJ 11199 were cultured in the same manner as described in Example 2, and the resultant culture broths were centrifuged to separate cells The cells were washed with physiological saline.

Twenty-five gram samples of the cells were suspended in 500 ml of 0 1 M phosphate buffer 5 solution (p H 8 0) which contained 5 g D-5-(p-hydroxyphenyl) hydantoin or 5 g L5-(p-hydroxyphenyl) hydantoin The reaction mixtures were held at 30 C for 27 hours Each mixture was centrifuged to separate the cells and filtered through an ultra-filtration membrane The p-hydroxyphenyl-glycine in these filtrates was adsorbed on the H: form of a cation exchange resin ("Diaion" SK-1 B) and was eluted with 1 N NH 4 OH 10 Crystals were obtained from each sample by cooling after evaporating the filtrates The crystals were purified by recrystallization in water-ethanol solution By the use of Achromobacter liquefaciens AJ 11198, 2 96 g of crystals were obtained from D5-(p-hydroxyphenyl) hydantoin and 3 12 g of crystals from L-5-(phydroxyphenyl) hydantoin When Alcaligenes aquamarinus AJ-11199 was used, 3 02 g of crystals were obtained 15 from D-5-(p-hydroxyphenyl) hydantoin and 3 06 g of crystals were obtained from L5-(p-hydroxyphenyl) hydantoin.

These crystals were shown to be the same as authentic D-phydroxyphenylglycine by correspondence of the NMR spectra, the Rf values of thin layer chromatograms and paper chromatograms and by optical rotations 20 Example 8

Moraxella nonliquefaciens AJ 1221, Paracoccus denitrificans AJ 11222 and Arthrobacter fragilus AJ 11223 were cultured in the same manner as described in Example 3, and the resultant culture broths were centrifuged to separate the cells The cells were washed with physiological saline 25 Twenty-five grams of the cells were suspended in 500 ml samples of 0 1 M phosphate buffer solution (p H 8 0) each of which contained 5 g of D-5-(phydroxyphenyl) hydantoin of g or L-5-(p-hydroxyphenyl) hydantoin Each reaction mixture was held at 30 C for 27 hours Each mixture was then centrifuged to separate the cells and then each solution was filtered with an ultra-filtration membrane The p-hydroxyphenylglycine in these filtrates was 30 adsorbed on the H form of a cation exchange resin ("Diaion" SK-1 B) and was eluted from the resin with 1 N NH 4 OH.

Crystals were obtained from each sample by cooling after evaporating the filtrates The crystals were purified by recrystallization in water-ethanol solution In the case of Moraxella nonliquefaciens AJ-11221, 4 09 g of crystals were obtained from D-5-(phydroxyphenyl) 35 hydantoin and 4 06 g from L-5-(p-hydroxy phenyl) hydantoin In the case of Paracoccus denitrificans AJ 11222, 2 82 g of crystals were obtained from D-5-(phydroxyphenyl) hydantoin and 2 86 g of crystal were obtained from L-5-(p-hydroxyphenyl) hydantoin In the case of Arthrobacter fragilus AJ 11223, 3 18 g of crystals were obtained from D5-(p-hydroxyphenyl) hydantoin and 3 10 g of crystals were obtained from L 40 5-(p-hydroxyphenyl) hydantoin.

These crystals were shown to be the same as authentic D-p-hydroxyphenylglycine by correspondence of the NMR spectra, the Rf values of thin layer chromatograms and paper chromatograms and by optical rotations.

Example 9 45

One loopful inoculum of Achromobacter liquefaciens AJ 11198 or Alcaligenes aquamarinus AJ 11199 which was previously cultured on a bouillonagar slant at 30 C for 24 hours was transferred to 50 ml of a culture medium placed in 500 ml flask The culture medium contained, per deciliter, 2 O g glucose, 0 5 g (NH 4)2 SO 4, 0 1 g KH 2 PO 4, 0 1 g K 2 HPO 4, 0 05 g Mg SO 4 7 H 20, lmg Fe SO 4 7 H 20, lmg Mn SO 4 4 H 20, 1 O g yeast extract, 50 0.2 g DL-5-cyanoethyl hydantoin, and Ca CO 3 (separately sterilized) The medium was adjusted to p H 7 0, and heated at 120 C for 15 minutes Cultivation was conducted at 30 C for 20 hours, and the cells thus obtained were collected by centrifugation, and washed with an equal volume of physiological saline.

The cells were suspended in 0 1 M phosphate buffer (p H 8 0) to a concentration of 5 g/dl 55 and contained lg/dl of 5-substituted hydantoin listed in Table 6 (the final volume of the buffer was 5 ml).

The amino acids formed in the reaction mixture were determined by the methods mentioned before, and ascertained as the D-isomer by measurement of optical rotation The results are shown in Table 6 60 1,587,116 Table 6

Amount of D-a-amino acid mg/m I 5-substituted hydantoin used D-a-amino acid produced AJ 11198 AJ 11199 DL-5-phenylhydantoin D-phenylglycine 3 21 3 83 DL-5-(p-hydroxyphenyl) hydantoinD-p-hydroxyphenylglycine 3 16 4 02 DL-5-(p-chlorophenyl) hydantoinD-p-chlorophenylglycine 1 82 1 96 DL-5-(p-methylphenyl) hydantoinD-p-methylphenylglycine 1 64 1 88 DL-5-(p-methoxyphenyl) hydantoinD-p-methoxyphenylglycine 3 18 4 04 Example 10

A fifty ml portion of a culture medium containing, per deciliter, 1 O g glucose, 0 5 g 10 (NH 4)2504, O lg KH 2 PO 4, 0 3 g K 2 HPO 4, O 05 g Mg SO 4 7 H 20, 1 mg Fe SO 4-7 H 20, lmg Mn SO 4 '4 H 2 O, 1 Og yeast extract and 0 2 g DL-5-methyl-mercaptoethyl hydantoin was placed in 500 ml flask and heated at 120 C for 15 minutes To the medium was added Sg/dl of Ca CO 3 whcih had been previously sterilized The culture medium was transferred with one loopful inocula of Pseudomonas caryophylli AJ 11150, which had been previously grown on 15 a bouillon-agar slant at 30 C for 24 hours, to the medium which was shaken at 30 After 16 hours of cultivation, DL-5-(p-hydroxyphenyl) hydantoin was added to the culture medium in a concentration of lg/dl Cultivation was conducted for an additional 24 hours In the resultant culture liquid, 5 83 mg/ml of D-p-hydroxyphenylglycine were found.

Example 11 20

A fifty ml portion of a culture medium containing, per deciliter, 2 O g glucose, O 5 g (NH 4)2 SO 4, O lg KH 2 PO 4, 0 3 g K 2 HPO 4, O 05 g Mg SO 4 7 H 20, lmg Fe SO 4 7 H 20, lmg Mn SO 4-4 H 20, 1 O g yeast extract, and 0 2 g DL-5-methylmercaptoethyl hydantoin at p H 7 0 placed in a 500 ml flask and sterilized at 120 C 15 minutes To the medium was added separately sterilized calcium carbonate 25 The medium was inoculated with Achromobacter liquefaciens AJ 11198 or Alcaligenes aquamarinus AJ 11199 which was previously cultured on a bouillonagar slant at 30 C for 24 hours, and shaken at 30 C.

After the cultivation had been continued for 16 hours, DL-5-(phydroxyphenyl) hydantoin was added to the culture medium in a concentration of lg/dl, and further cultivation was 30 performed for 24 hours Analyses of the resultant culture liquids showed that 0 56 g/dl and 0.58 g/dlof D-p-hydroxyphenylglycine accumulatedfor AJ 11199 respectively.

Example 12

The cells of Pseudomonas caryophylli AJ 11150 prepared by the method as described in Example 1 were suspended in 0 1 M phosphate buffer in a concentration of 5 g/dl (final 35 volume being 10 ml) The medium was treated with 20 KC supersonic waves for 5 minutes.

Then, to 5 ml of the supernatant of the suspension obtained by centrifugation was added DL-5-(p-hydroxyphenyl) hydantoin in a resulting concentration of lg/dl The solution was adjusted to p H 8 The reaction mixture was held at 30 C for 24 hours, and 7 20 mng/ml D-p-hydroxyphenylglycine were found in the reaction mixture 40 Example 13

In the method shown in Example 12, the cells of Achromobacter liquefaciens AJ 11198 or Alcaligenes aquamarinus both prepared by the method shown in Example 2 were used in place of the cells of AJ 11150.

AJ 11198 produced O 64 g/dl of amino acidproduct in the reaction mixture, and AJ 11199 45 produced 0 68 g/dl of the same amino acid product.

Example 14

In the method shown in Example 12, the cells of Moraxella nonliquefacie AJ 11221, Paracoccus denitrificans AJ 11222 or Arthrobacter fragilus AJ 11223 prepared by themethod shown in Example 3, were used In the resultant reaction mixture, AJ 11221, AJ 50 11222 and AJ 11223 were produced to yield 0 82 g/dl 0 51 g/dl and 0 67 g/dl of Dp-hydroxyphenylglycine, respectively.

Example 15

A one gram amount of the cells of Pseudomonas caryophylli AJ 11150 prepared by the method shown in Example 1 was suspended in 4 ml of deionized water and the suspension 55 was cooled To the suspension was added 750 mg acrylamide and 45 mg methylenebisacrylamide Nitrogen gas was introduced into the suspension to remove oxygen gas from the suspension, and thereafter 3 5 mg of ammonium persulfate and 8,u 1 of N,N'dimethylaminopropionitrile was added to the suspension and cooled for 1 hour The gel thus formed was passed through a 50 mesh wire gauze, and 2 g of the gel was put into 5 ml of 60 0.1 M phosphate buffer (p H 8 0) containing lg/dl of DL-5-(phydroxyphenyl) hydantoin.

The reaction mixture was then held at 30 C for 24 hours, and 3 24 mg/ml of Dp-hydroxyphenylglycine was found in the reaction mixture.

By a procedure analogous to that mentioned above, Achromobacter liquefaciens AJ 11198,Alcaligenesaquamarinus AJ 11199,Moraxellanonliquefaciens AJ 11221, Paracoc 65 1597 1 1 6 cus denitrificans AJ 11222 and Arthrobacterfragilus AJ 11223 led to the production of 0 34 g/dl, 0 35 g/dl, 0 50 g/dl, 0 28 g/dl, and 0 38 g/dl D-phydroxyphenylglycine, respectively in the reaction mixture.

Example 16

An aqueous culture medium containing, per deciliter, 2 O g glucose, O 5 g (NH 4)2 SO 4, O lg 5 KH 2 PO 4, 0 1 g K 2 HPO 4, O 05 g Mg SO 4 7 H 20, lmg Fe SO 4-7 H 20, lmg Mn SO 44 H 20, 1 Og yeast extract and 4 O g Ca CO 3 was adjusted to p H 7 0, and 50 ml batches of the culture medium were placed in 500 ml flasks which were heated at 120 C for 15 minutes.

Pseudomonas caryophylli AJ 11150 previously cultured on a bouillon-agar slant at 30 C for 24 hours was inoculated into the culture medium mentioned above, and cultured at 30 C 1 O After 16 hours of cultivation the 5-substituted hydantoins shown in Table 7 which had been previously heated to sterilize the same were placed in samples of the culture medium at a concentration of O 2 g/dl and additional cultivation was performed for 6 hours Cells in the culture liquids thus obtained were collected by centrifugation, and washed with the physiological saline 15 Five ml samples of 0 1 M phosphate buffer (p H 8 0) containing lg/dl DL-phydroxyphenyl hydantoin and Sg/dl of the cells mentioned above were helda 30 C for 16 hours The amounts of D-p-hydroxyphenylglycine produced in each reaction mixture are shown in Table 7.

Table 7 20

5-substituted hydantoin D-p-OH-phenylglycine added to the culture medium produced (mg/ml) none 0 92 hydantoin 0 98 DL-5-methyl hydantoin 1 72 DL-5-isopropyl hydantoin 2 35 DL-5-isobutylhydantoin 2 04 25 DL-5-( 1 '-methylpropyl) hydantoin 1 82 DL-5-hydroxymethyl hydantoin 1 96 DL-5-(l'-hydroxyethyl)hydantoin 2 31 DL-5-methylmercaptoethyl hydantoin 5 21 DL-5-carbamoylmethyl hydantoin 1 99 DL-5-carbamoylethyl hydantoin 2 16 DL-5-benzyl hydantoin 3 99 30 DL-5-(p-hydroxybenzyl) hydantoin 2 04 DL-5-indolylmethyl hydantoin 5 06 DL-5-( 5 '-hydroxyindolylmethyl) hydantoin 2 03 DL-5-carboxymethyl hydantoin 1 92 DL-5-carboxyethyl hydantoin 1 78 DL-5-( 4 '-imnidazoylmethyl) hydantoin 1 72 DL-54 '-aminobutyl)hydantoin 1 99 35 DL-5-( 3 '-guanidpropyl) hydantoin 2 03 DL-5-( 3 '-aminopropyl) hydantoin 1 92 DL-5-( 3 '-ureidpropyl) hydantoin 1 70 DL-5-( 3 '-sulfhydrylethyl) hydantoin 2 06 DL-5-phenyl hydantoin 3 99 DL-5-(p-hydroxyphenyl) hydantoin 4 72 DL-5 p-chlorophenyl)hydantoin 4 19 40 DL-5p-methylphenyl) hydantoin 4 00 DL-5p-methoxyphenyl) hydantoin 4 92 DL-5p-benzyloxyphenyl) hydantoin 2 42 DL-5 3 ' 4 '-dihydroxybenzyl) hydantoin 2 88 DL-5 3 '4 '-dimethoxybenzyl) hydantoin 2 46 DL-5 3 ' 4 '-methylenedioxy) hydantoin 2 72 DL-5-cyanoethyl hydantoin 5 42 45 Example 17

Two 300 ml batches of a culture medium containing, per deciliter, 2 g glucose, O 5 g (NH 4)2504, 0 1 g KH 2 PO 4, 0 02 g Ca C 12-2 H 20, 0 3 g K 2 HPO 4, O 05 g Mg SO 47 H 20, lmg Fe SO 4 7 H 20, lmg Mn SO 4 4 H 20, and 1 O g yeast extract at p H 7 0 were placed in 1 liter fermentation vessels The vesselswere heated at 1200 C for 15 minutesto sterilize the same 50 Six ml samples of seed cultures of Moraxella nonliquefaciens AJ 11221 and Pseudomonas caryophylli AJ 11150 which had been previously cultured at 30 C for 24 hours in the same medium mentioned above were transferred into fermentation vessels Cultivation was conducted under aerobic conditions at 30 C at an adjusted p H of 7 0 with gaseous ammonia.

After 16 hours and 22 hour of cultivation, DL-5-(p-hydroxyphenyl) hydantoin samples were 55 added to each culture medium to achieve a concentration of 0 2 g/dl.

After 25 hours from initiation of cultivation, nitrogen gas was introduced into the fermentation vessels, and the temperature was raised to 40 C Then, crystals of DL-5-(phydroxyphenyl) hydantoin were added to each culture medium to a concentration of 3 g/dl.

Each culture medium was held for 40 hours at 40 C while nitrogen was continually intro 60 duced into each flask The p H was adjusted to 7 0 with 1 N HCI AJ 11221 produced 2 75 g/dl of D-p-hydroxyphenylglycine and AJ 11150 produced 2 69 g/dl of the same.

Claims (1)

1. WHAT WE CLAIM IS:

   1 A process for producing a D-ac-amino acid, which comprises contacting in an aqueous medium at a p H in the range from 4 to 9 a 5-substituted hydantoin with an effective amount of 65 1 587 116 25 an enzyme capable of converting the 5-substituted hydantoin to the D-aamino acid and produced by a microorganism, the microorganism being capable of utilizing the D-isomer of the 5-substituted hydantoin as the sole nitrogen source, but substantially incapable of utilizing the L-isomer of the 5-substituted hydantoin as the nitrogen source, and the substituent at the 5-position being such that upon reaction with the enzyme an optically active 5 D-a-amino acid isomer is produced.

   2 A process according to claim 1, which also includes recovering the D-aamino acid which accumulates in the aqueous medium.

   3 A process according to claim 1 or 2, wherein the microorganism belongs to the genus Pseudomonas 10 4 A process according to claim 3, wherein the microorganism is Pseudomonas solanacearum AJ 11149 (NRRL B 11,255), Pseudomonas caryophilli AJ 11150 (NRRL B 11,256), Pseudomonas diminuta AJ 11151 (NRRLB-11,257), or Pseudomonas diminuta AJ 11152 (NRRL B-11,258) 15 A process according to claim 1 or 2, wherein the microorganism belongs to the genus Achromobacter or Alcaligenes.

   6 A process according to claim 5, wherein the microorganism is Achromobacter liquefaciens AJ 11198 (NRRLB-11,259 or Alcaligenes aquamarinus AJ 11199 (NRRL B11,260).

   7 A process according to claim 1 or 2, wherein the microorganism belongs the the genus 20 Moraxella, Paracoccus or Arthrobacter.

   8 A process according to claim 7, wherein the microorganism is Moraxella nonliquefaciens AJ 11221 (NRRLB-11,261), Paracoccus denitrificans AJ-11222 (NRRL B-11,262), or Arthrobacterfragilus AJ 11223 (NRRL B-11,263) 25 9 A process according to any preceding claim, wherein the enzyme is produced by culturing the microorganism in an aqueous culture medium containing at least one 5-substituted hydantoin.

   A process according to any preceding claim, wherein the 5-substitutedhydantoin contacted by the enzyme is a 5-alkyl hydantoin 30 11 A process according to any one of claims 1 to 9, wherein the 5substituted-hydantoin contacted by the enzyme is a 5-(sub-substiuted alkyl) alkyl) hydantoin.

   12 A process according to any one of claims 1 to 9, wherein the 5substituted-hydantoin contacted by the enzyme is a 5-arylhydantoin.

   13 A process according to any one of claims 1 to 9, wherein the 5substituted-hydantoin 35 contacted by the enzyme is a 5-(substituted-aryl)-hydantoin.

   14 A process according to any one of claims 1 to 9 wherein the 5substituted-hydantoin contacted by the enzyme is a 5-aralkyl hydantoin.

   A process according to any one of claims 1 to 9, wherein the 5substituted-hydantoin contacted by the enzyme is a 5-(substituted aralkyl) hydantoin 40 16 A process according to any of claims 1 to 9, wherein the 5-substituted hydantoin contacted by the enzyme is 5-phenyl hydantoin or 5-p-hydroxyphenyl hydantoin.

   17 A process according to any one of claims 1 to 5, wherein the 5substituent of said hydantoin contacted by the enzyme is a straight, branched or cyclic saturated aliphatic hydrocarbon radial; a straight, branched or cyclic unsaturated aliphatic hydrocarbon radical; 45 a straight, branched or cyclic saturated or unsaturated aliphatic hydrocarbon radical of which at least one hydrogen atom is substituted with at least one further substituent; an aromatic hydrocarbon radical; an aromatic hydrocarbon radical of which at least one hydrogen atom is substituted with at least one further substituent; a heterocyclic radical or a heterocyclic radical of which at least one hydrogen atom is substituted with at least one further sub 50 stituent.

   18 A process according to any preceding claim, wherein the 5-substitutedhydantoin contacted by the enzyme is contacted with the enzyme ns the aqueous culture medium in which the microorganism is cultured.

   19 A process according to any one of claims 1 to 17, wherein the 5 55 substituted-hydantoin is contacted with the enzyme in an aqueous reaction medium containing as the enzyme source the cells of the microorganism, the homogenate of the cells, cells which have been treated with supersonic waves, freeze-dried cells, cells dried by treatment with an organic solvent, protein fractions separated from cells which have the enzyme activity, or immobilized cells 60 A process according to claim 1, substantially as described in the foregoing Example 1.

   21 A process according to claim 1, substantially as described in the foregoing Example 2.

   22 A process according to claim 1, substantially as described in the foregoing Example 3.

   23 A process according to claim 1, substantially as described in the foregoing Example 4.

   24 A process according to claim 1, substantially as described in the foregoing Example 5 65 1.587,116 A process according to claim 1, substantially as described in the foregoing Example 6.

   26 A process according to claim 1, substantially as described in the foregoing Example 7.

   27 A process according to claim 1, substantially as described in the foregoing Example 8.

   28 A process according to claim 1, substantially as described in the foregoing Example 9.

   29 A process according to claim 1, substantially as described in the foregoing Example 5 10.

   A process according to claim 1, substantially as described in the foregoing Example 11.

   31 A process according to claim 1, substantially as described in the foregoing Example 12 10 32 A process according to claim 1, substantially as described in the foregoing Example 13.

   33 A process according to claim 1, substantially as described in the foregoing Example 14.

   34 A process according to claim 1, substantially as described in the foregoing Example 15 15.

   A process according to claim 1, substantially as described in the foregoing Example 16.

   36 A D-a-amino acid whenever produced by a process according to any preceding claim 20 For the Applicants HASELTINE LAKE & CO.

   Chartered Patent Agents 28 Southampton Buildings Chancery Lane 25 London WC 2 A 1 AT.

   and Temple Gate House Temple Gate Bristol B 51 6 PT 30 Printed for Her Majesty's Stationery Office, by Croydon Printing Company Limited, Croydon, Surrey, 1981.

   Published by The Patent Office 25 Southampton Buildings London, WC 2 A LAY, from which copies may be obtained.